The overall research objective is to characterize the metabolic processes occurring in Salmonella which contribute to its survival in animal hosts and its pathogenicity. Because intestinal infection is an anaerobic phenomenon, an understanding of salmonellosis necessarily involves studying anaerobic metabolism. Here the experimental focus is on an anaerobic activity performed by Salmonella species, but not by Escherichia coli, namely the production by hydrogen sulfide from thiosulfate and sulfite. The proposed research for this grant period is designed to characterize hydrogen sulfide production by Salmonella typhimurium from both the biochemical and genetic standpoints and to evalute the physiological role of this pathway including its contribution to pathogenicity. The reductases which catalyze the formation of hydrogen sulfide will be purified and characterized with respect to structural and catalytic parameters, electron donors, and electron carriers. Extensive mutant analysis is planned in order to identify the genes involved in hydrogen sulfide production and the associated mechanisms of regulation. Insertion mutants [both TH10 and Mu d(lac)] and point mutants defective in hydrogen sulfide production will be isolated, characterized, and mapped. Double and triple mutants will regulatory alterations in addition to the primary defects in hydrogen sulfide production will also be characterized; these studies will facilitate the identification of the different levels of regulation, including regulation by fnr and by nitrate. The ability of thiosulfate and sulfite to serve as electron acceptors for growth on non-fermentable carbon sources will be examined by means of growth studies and presence of competitors (specifically Escherichia coli) will also be examined. Finally, the importance of hydrogen sulfide production to pathogenicity will be tested by challenging mice with appropriate mutant strains defective in hydrogen sulfide production.